Recent studies have revealed that the maintenance and retention of hematopoietic stem cells (HSCs) in the bone marrow (BM) are dependent upon extrinsic signals from cells within the BM microenvironment or niche, including vascular endothelial cells (ECs), perivascular cells, osteolineage cells, mesenchymal stromal cells and sympathetic neurons. However, the precise mechanisms through which niche cells individually or in concert regulate HSC homeostasis have yet to be fully elucidated. Furthermore, the mechanisms controlling HSC regeneration, which is necessary for the recovery of the hematopoietic system following myelosuppressive chemo- and radiotherapy, remain less well understood. Here, we will use innovative mouse models and molecular techniques to define the mechanism through which the novel secreted protein, pleiotrophin (PTN), regulates HSC fate in vivo and to identify the BM niche cells responsible for PTN-mediated maintenance and regeneration of the HSC pool. Differential gene expression analysis of HSC-supportive ECs revealed the overexpression of PTN, a heparin binding growth factor, which is normally expressed in the hippocampus and functions as a neurite outgrowth factor. Short-term culture of murine BM HSCs with PTN caused more than 10-fold expansion of long-term repopulating HSCs, whereas deletion of PTN in the BM microenvironment also caused a 10-fold deficit in HSC content in mice. Anatomic studies suggest that PTN is expressed by VEGFR3+VEcadherin+ sinusoidal ECs and CXCL12+ perivascular cells in the BM, suggesting that PTN is a unique component of the HSC vascular niche. In preliminary studies, PTN-mediated expansion of hematopoietic stem/progenitor cells in vitro was abrogated by pharmacologic inhibitors of Ras or MEK, suggesting that PTN mediates HSC growth via induction of the Ras/MEK signaling pathway. Based upon these findings, we hypothesize that PTN is a paracrine regulator of the HSC pool within the BM vascular niche and regulates HSC growth via activation of the Ras/MEK/ERK pathway. In keeping with this hypothesis, we propose the following Specific Aims: I) Determine the role of the Ras/MEK/ERK pathway in mediating PTN effects on HSCs, 2) Determine which HSC niche cells are responsible for mediating PTN effects on HSC maintenance and regeneration in vivo, and 3) Determine whether PTN can expand human HSCs ex vivo or in vivo via activation of Ras/MEK signaling pathway. Our broad, long-term objective will be to define the mechanisms through which PTN mediates the self-renewal and regeneration of HSCs to facilitate translation of PTN for the therapeutic expansion of human HSCs.